This SPIRAT proposal aims to develop and clinically evaluate gene therapy for HIV infection. This project will focus on the development and comparison of murine retrovirus, HIV and AAV vectors for delivery of ribozymes and other anti-HIV genes into primary hematopoietic cells, although integration of AAV genome still requires cell division. HIV vectors have the further advantage of being activated and rescued by HIV infection, as well as tracking their RNA transcripts with HIV RNA intracellularly. Through close interaction with the other projects, we hope to develop the next generation of ribozyme vectors for HIV infection, which can be ready for clinical evaluation within the term of this SPIRAT award. The ultimate goal of our efforts is to develop vectors for in vivo delivery. The specific aims of this project are: (1) Development of AAV vector for gene delivery and assessment of efficacy-Efforts will be directed at achieving efficient transduction of primary human cells, including unstimulated human PBL, terminally differentiated macrophage/monocytes and CD34+ stem cells in the absence and presence of growth factors. The transduced cells will be assessed for the persistence of gene expression, AAV integration and degree of protection from challenge by HIV infection. (2) Development of HIV vectors for gene delivery-Efforts will include optimization of packaging cell lines and vectors derived from the dual tropic viruses HIV-1(MN) and HIV-2(KR). VSV-G pseudotypes of HIV vectors will also be generated. The efficiency of transduction of primary PBL by vectors delivering ribozymes or other anti-HIV genes will be determined. (3) Comparison of gene delivery by AAV vs. retroviral vectors into T-cell lines, human PBL, human macrophages/monocytes and CD34+ stem sells.